PROJECT SUMMARY Oral squamous cell carcinoma (OSCC) claims a quarter of a million lives each year. Epidemiological evidence has shown that metformin, the most commonly prescribed therapy for type 2 diabetes mellitus, is correlated with a reduced risk of carcinogenesis. Strong evidence from murine models and in vitro studies suggest that metformin can also reduce OSCC growth, even in the context of metabolic health. Therefore, a phase II clinical trial (M4OC- Prevent, NCT02581137) was initiated to test the potential of metformin to reduce the pathology of oral premalignant lesions (OPL) in non-diabetic patients. Preliminary analysis of this trial has shown that acute metformin treatment induces a remarkable reduction in pathology. However, the mechanism underlying metformin?s anti-neoplastic properties remains unknown. Interestingly, over the past year multiple studies have implicated microbial dysbiosis in cancer onset and therapy. Independently, the anti-diabetic properties of metformin were shown to be caused by dramatically altering the gut microbiome. Earlier this year, a large prospective study found significant differences in the oral microbiome that were predictive of future OSCC development. However, to date no studies have examined the response of the oral microbiome to metformin treatment. In this proposal, we will test the hypothesis that metformin affects the oral microbiome and that this effect is related to the anti-neoplastic properties of metformin. Unstimulated saliva samples from patients enrolled in clinical trial M4OC- Prevent, NCT02581137 were collected before and after metformin treatment, and the microbiome will be evaluated using novel, quantitative microbial analysis technologies developed in our lab. Additionally, the diurnal dynamics of the oral microbiome in healthy individuals will be evaluated in a controlled experiment to determine potential sources of bias in our patient cohort. Lastly, in vitro culturing and metabolomics experiments taking advantage of genetically modified OSCC cells will allow us to determine mechanistic background and identify molecules relevant for this relationship. The results from these experiments will greatly enhance our knowledge of the saliva microbiome and have the potential to dramatically increase our understanding of human oral health.